Milk protein-derived peptides induce 5-HT2C-mediated satiety in vivo

International Dairy Journal 38 (2014) 55e64

Contents lists available at ScienceDirect

International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj

Milk protein-derived peptides induce 5-HT2C-mediated satiety in vivo Harriët Schellekens a, *,1, Alice B. Nongonierma b,1, Gerard Clarke c, e, Wesley E.P.A. van Oeffelen f, Richard J. FitzGerald b, Timothy G. Dinan a, c, e, John F. Cryan a, d, e a

Food for Health Ireland, University College Cork, Co. Cork, Ireland Department of Life Sciences and Food for Health Ireland, University of Limerick, Castletroy, Limerick, Ireland c Department of Psychiatry, University College Cork, Co. Cork, Ireland d Department of Anatomy and Neuroscience, University College Cork, Co. Cork, Ireland e Laboratory of Neurogastroenterology, Alimentary Pharmabiotic Centre, University College Cork, Co. Cork, Ireland f School of Pharmacy, University College Cork, Co. Cork, Ireland b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 October 2013 Received in revised form 11 April 2014 Accepted 14 April 2014

This study investigates the ability of milk protein-derived peptides to specifically activate the serotonin 2C (5-HT2C) receptor, a key receptor in central regulation of food intake. A dose dependent 5-HT2C receptor activation by the 1 kDa ultrafiltration permeates of a sodium caseinate (NaCNH-1 kDa permeate) and a whey protein hydrolysate (WPH-1 kDa permeate) was demonstrated using an intracellular calcium mobilisation assay in human embryonic kidney (Hek) cells expressing the 5-HT2C receptor. Both samples activated the 5-HT2C but not the 5-HT2A and 5-HT2B receptors. NaCNH-1 kDa permeate significantly (p < 0.01) reduced cumulative food intake when administered to male mice (C57Bl/6) by intraperitoneal injection at 500 mg kg
1 body weight. In contrast, no effect of WPH-1 kDa permeate could be seen on food intake in vivo. These results demonstrate the promising appetite-suppressing potential of NaCNderived peptides, targeting the 5-HT2C receptor. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction As the incidence of obesity and its co-morbidities are increasing worldwide, so does the need for novel anti-obesity intervention strategies (Bloom et al., 2008; Nguyen et al., 2012). Current anti-obesity treatments consist of dietary and lifestyle interventions as well as pharmaceutical drug treatments and surgery (Aronne, Powell, & Apovian, 2011; Cecchini et al., 2010; Derosa & Maffioli, 2012; Halford, Boyland, Blundell, Kirkham, & Harrold, 2010; Kang & Park, 2012; Powell, Apovian, & Aronne, 2011; Powell & Khera, 2010; Schellekens, Dinan, & Cryan, 2009). However, most pharmaceutical drug candidates have so far failed to reach the market, due to their low potency and efficacy or side effects and safety concerns (Aronne et al., 2011; Chakrabarti, 2009; Derosa & Maffioli, 2012; Kang & Park, 2012; Kennett & Clifton, 2010). Moreover, diets are generally associated with a low

* Corresponding author. Tel.: þ353 21 4901771. E-mail address: [email protected] (H. Schellekens). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.idairyj.2014.04.004 0958-6946/Ó 2014 Elsevier Ltd. All rights reserved.

success rate due to poor adherence and compliance. Bioactive peptides from natural sources may have potential as weight management ingredients and are becoming of considerable interest in the food industry to counteract obesity when incorporated into the diet (Gonzalez-Castejon & Rodriguez-Casado, 2011; Gooda Sahib et al., 2012). Weight loss can be achieved following the modulation of appetite and satiety signalling pathways in the central nervous system (CNS) (Obici, 2009; Valentino, Lin, & Waldman, 2010). Modulation of the central serotonergic system, in particular activation of the centrally expressed serotonin (5-hydroxytryptamine, 5-HT) 2C receptor (5-HT2C), stimulates satiety via excitatory neurotransmission. Therefore, activation of the 5-HT2C receptor is a target for anti-obesity strategies (Dutton & Barnes, 2006; Garfield & Heisler, 2009; Lam et al., 2008; Miller, 2005; Schellekens, Clarke, Jeffery, Dinan, & Cryan, 2012; Somerville, Horwood, Lee, Kennett, & Clifton, 2007; Tecott, 2007; Tecott et al., 1995; Vickers, Clifton, Dourish, & Tecott, 1999). Several drugs targeting the central serotonergic system, such as sibutramine, and fenfluramine, have been specifically developed to induce satiety, or as for m-chlorophenylpiperazine (mCPP), found to reduce food intake as a secondary effect (Dalton, Lee, Kennett, Dourish, & Clifton, 2004, 2006;

56

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

Halford, Harrold, Boyland, Lawton, & Blundell, 2007; Vickers et al., 1999). However, these drugs have been associated with poor efficacy and safety concerns due to heart and pulmonary vasculature side-effects and non-specific effects (Miller, 2005). Recently, the specific 5-HT2C receptor agonist, Lorcaserin, was approved for the treatment of obesity (Jandacek, 2005; Martin et al., 2011; O’Neil et al., 2012; Redman & Ravussin, 2010). This highlights the benefits of targeting the 5-HT2C receptor in weight management strategies. Natural compounds, including milk protein-derived bioactive peptides, are presumed to be safe because they are derived from food sources. In addition, they have not been associated with the undesirable side effects, which are often encountered with pharmaceutical drugs. Milk proteins represent a unique source of biologically active peptides which have been associated with various health benefits (FitzGerald, Murray, & Walsh, 2004; Korhonen, 2009; Kreider et al., 2011; Lonnerdal, 2003; Meisel & FitzGerald, 2003; Nagpal et al., 2011; Phelan & Kerins, 2011). These include anti-hypertensive, mineral binding, antimicrobial, immuno-regulatory, antidiabetic, antioxidant and opioid peptides (Booij, Merens, Markus, & Van der Does, 2006; Froetschel, Azain, Edwards, Barb, & Amos, 2001; Korhonen, Marnila, & Gill, 2000; Kumar et al., 2012; Nongonierma & FitzGerald, 2012a, 2013; Orosco et al., 2004; Power, Jakeman, & FitzGerald, 2013; Virtanen, Pihlanto, Akkanen, & Korhonen, 2007). In particular, the satiating effects of milk proteins and milk protein-derived peptides may play a role in appetite regulation, weight loss and/or prevention of weight gain (Anderson, Tecimer, Shah, & Zafar, 2004; Burton-Freeman, 2008; Dougkas, Reynolds, Givens, Elwood, & Minihane, 2011; Luhovyy, Akhavan, & Anderson, 2007; van Meijl, Vrolix, & Mensink, 2008; Pilvi, Korpela, Huttunen, Vapaatalo, & Mervaala, 2007; Pilvi et al., 2008; Ricci-Cabello, Herrera, & Artacho, 2012; Staljanssens et al., 2012; Veldhorst et al., 2009; Zemel, 2005). It has been demonstrated that diets rich in a-lactalbumin could enhance 5-HT release in rats (Orosco et al., 2004) and in humans (Booij et al., 2006; Nieuwenhuizen et al., 2009). This was attributed to a higher bioavailability of tryptophan, the precursor of 5-HT. In addition, the milk protein-derived bioactive peptide,

b-casomorphin-7 (TyreProePheeValeGlueProeIle), has been shown to bind and antagonise 5-HT2 receptors (Sokolov et al., 2005). Recently, it was demonstrated that milk protein hydrolysates generated from different starting substrates, including sodium caseinate (NaCN), acid casein, skim milk powder and glycomacropeptide, behave as 5-HT2C receptor agonists in vitro (Nongonierma, Schellekens, Dinan, Cryan, & Fitzgerald, 2013). The aim of this study was to assess the role of NaCN-derived and whey protein (WP)-derived bioactive peptides on 5-HT2C receptor agonism. In addition, the specificity of NaCN and WP-derived bioactive peptides for the 5-HT2C receptor over the closely related 5-HT2A and 5-HT2B receptors was studied. Finally, the effect of NaCN and WP hydrolysates on cumulative food intake in mice was assessed as a measure of the satiety inducing potential of these milk proteinderived peptides in vivo. Milk protein-derived bioactive peptides with 5-HT2C receptor agonist properties may have considerable potential in the development of non-pharmacological weight management ingredients and may be exploited in the development of anti-obesity functional foods. 2. Materials and methods 2.1. Generation of the milk protein hydrolysates and their associated ultrafiltration fractions The experimental design of this study is summarised in Fig. 1. Sodium caseinate (NaCN; 90.4%, w/w, protein) was provided by Kerry Ingredients (Listowel, Co. Kerry, Ireland) and whey proteins (WP; 88.3%, w/w, protein) were from Carbery Milk Products (Ballineen, Co. Cork, Ireland). The 240 min NaCN hydrolysate (NaCNH) was produced by enzymatic hydrolysis as described by Nongonierma et al. (2013). The whey protein hydrolysates (WPH) were generated using similar enzymatic hydrolysis conditions as for the NaCNH. Briefly, an aqueous solution of WP (10%, w/v, on a protein basis) was rehydrated for 1 h at 50  C with gentle mixing. The pH was adjusted to 3.0 with 1.0 M HCl before addition of pepsin. The pH of the reaction mixture was maintained constant

Fig. 1. Schematic representation of the experimental design including the enzymatic hydrolysis, ultrafiltration fractionation and bioassay testing of the 1 kDa permeates of the 240 min sodium caseinate (NaCNH-1 kDa permeate) and whey protein hydrolysates (WPH-1 kDa permeate). Abbreviations are: NaCN, sodium caseinate; NaCNH, 240 min sodium caseinate hydrolysate; WP, whey proteins; WPH, 240 min whey protein hydrolysate; RP-UPLC, reverse-phase ultra-performance liquid chromatography; GP-HPLC, gel permeationhigh performance liquid chromatography; Hek, human embryonic cells; 5HT2C, serotonin 2C receptor.

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

throughout hydrolysis using a pH stat titrator (Titrando 843, Tiamo 1.4 Metrohm, Dublin, Ireland) for 4 h. In addition, aliquots of the WPH were withdrawn at 10, 30, 60, 120 min throughout the hydrolysis reaction. WPH was generated in two independent duplicates (n ¼ 2). The enzyme was heat inactivated at 90  C for 20 min. The 4 h WPH was further fractionated by ultrafiltration (UF) using a cross-flow ultrafiltration unit (Sartoflow Alpha filtration System, Sartorius, Göttingen, Germany). WPH was sequentially UF fractionated through 10, 5 and 1 kDa cut-off stabilised cellulose UF cassettes (Sartorius). Ultrafiltration was carried out at 30  C. Six fractions of WPH were collected (10, 5 and 1 kDa permeate and retentate). The samples were freeze-dried (FreeZone 18L, Labconco, Kansas City, U.S.A.) and stored at
20  C until utilisation. 2.2. Reverse phase-ultra-performance liquid chromatography and gel permeation-high performance liquid chromatography analysis of WPH and its associated UF fractions Unhydrolysed whey proteins (WP), WPH and its UF fractions were analysed with an Acquity ultra-performance liquid chromatograph (Acquity UPLC; Waters, Dublin, Ireland) as described by Nongonierma and FitzGerald (2012b). Samples were resuspended at 0.5% (w/v) in 0.1% (v/v) trifluoroacetic acid (TFA, SigmaeAldrich, Dublin, Ireland) in high performance liquid chromatography (HPLC) grade water (VWR, Dublin, Ireland). Separation of peptides and individual milk proteins was carried out at 30  C using a 2.1  50 mm, 1.7 mm Acquity UPLC C18 BEH column mounted with a 0.2 mm inline filter (Waters). The absorbance of the eluant was monitored at 214 nm. The molecular mass profiles of the proteins and peptides present in the samples was determined by gel permeation GP-HPLC as described by Spellman, O’Cuinn, and FitzGerald (2009). Samples were resuspended at 0.25% (w/v) in 0.1% TFA and 30% HPLC grade acetonitrile (VWR) in HPLC grade water. A 600  7.5 mm I.D. TSK G2000 SW column mounted with a 75  7.5 mm I.D. TSKGEL SW guard column (Tosoh Bioscience, Stuttgart, Germany) was used for the separation. The absorbance was monitored at 214 nm. 2.3. Cell culture, in vitro transfection and lentiviral transduction Human embryonic kidney cells (Hek293A) were cultured and maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM; SigmaeAldrich) supplemented with 10% (v/v) heat inactivated foetal bovine serum (FBS; SigmaeAldrich) and 1% (v/v) GibcoÒ MEM non-essential amino acids (NEAA; Life technologies, Biosciences, Dublin, Ireland) in an atmosphere of 95% air and 5% CO2 at 37  C. Stably transfected Hek293A cells were maintained in complete medium supplemented with 300 ng mL
1 geneticin (G418; Merck, Darmstadt, Germany) as maintenance antibiotic. Hek293A cells stably expressing the unedited 5-HT2C-INI or the partly edited 5-HT2C-VSV isoform of the 5-HT2C receptor were generated as described by Schellekens, van Oeffelen, Dinan, and Cryan (2013). Briefly, Hek293A cells were transfected with plasmid construct expressing the unedited INI (H3309; Accession code, NM_000868; Genecopeia, MD, USA) or the partly edited VSV isoform of the 5-HT2C receptor (T0336; Genecopeia. Accession code: AF208053.1) with a C-terminal-EGFP tag, from a CMV promoter using lipofectamineÒ LTX plus reagent from InvitrogenÔ (Life technologies, Biosciences), according to the manufacturer’s instructions. The 5-HT2A receptor was amplified from the commercial construct 3  HA-tagged (N-terminus) 5-HT2A (HTR02ATN00, Missouri S&T cDNA Resource Center, MO, USA) and cloned into the pEGFP-N1 expression vector (6085-1, Clontech, Saint-Germain-en-Laye, France). Cells stably expressing the insert gene-EGFP fusion proteins were selected using flow assisted cell

57

sorting (FACS, Becton Dickinson FACSVantage SE, Oxford, UK) and G418 as a selection antibiotic. Hek293A cells expressing the 5-HT2B receptor were generated using lentiviral transductions, using a third generation packaging, gene delivery and viral vector production system developed by Follenzi and Naldini (2002a,b), Naldini (1998), Naldini et al. (1996), Vigna and Naldini (2000). The 5-HT2B receptor (Missouri S&T; HTR02BTN00) construct was cloned into the lentiviral expression vector, pHR-SIN-BX-EGFP, modified from the lentiviral expression plasmid pHR-SIN-BXIRES-EmGFP vector (gift of Adrian Thrasher, Institute of Child Health, London, UK). The generated HIV-based lentiviral vector (lv) particles, pseudotyped with the vesicular stomatitis virus G (VSVG), expressing the G-protein coupled receptor (GPCR) constructs from a spleen focus-forming virus (SFFV) promoter were produced using 293T-17 cells, following transient cotransfection of the cloned expression construct, pHR-5-HT2B-EGFP, the packaging construct, pCMVDR8.91 and the envelope construct, pMD.GeVSVG. Cells were transduced with lv expressing the lv5-HT2B-EGFP receptor diluted in transduction media, consisting of DMEM with 2% (v/v) heatinactivated FBS, 1% (v/v) NEAA and an additional 8 mg mL
1 polybreneÒ (H9268, SigmaeAldrich). Fluorescence was monitored using flow cytometry (BD FACS Calibur, BD Biosciences) as an indicator of receptor expression. 2.4. Calcium mobilisation assay Receptor mediated changes in intracellular Ca2þ were monitored on a Flex station II multiplate fluorometer (Molecular Devices Corporation, Sunnyvale, CA, USA). Stably transfected cells were seeded in sterile black-wall and clear flat bottom 96-well microtitre plates (3904, Costar, Fisher Scientific, Dublin, Ireland) at a density of 2.5  105 cells mL
1 (i.e., 2.5  104 cells per well) and maintained for w24 h at 37  C in a humidified atmosphere containing 5% CO2. After removal of the growth medium, cells were incubated with 25 mL of assay buffer (1 Hank’s buffered salt solution (HBSS) supplemented with 20 mM HEPES buffer) and 25 mL of Ca2þ 4 dye (R8141, Molecular Devices Corporation) according to the manufacturer’s protocol. FBS (SigmaeAldrich) and 5-HT (H9523; Sigmae Aldrich) diluted in assay buffer were used as controls. Milk protein hydrolysates were resuspended in assay buffer (1 HBSS supplemented with 20 mM HEPES buffer). Suspensions were centrifuged at 730  g (Jouan C3i multifunction centrifuge, Thermo Scientific, Dublin, Ireland) for 5 min at 4  C and filtered through 0.45 mm low protein binding filters (Sartorius, Dublin, Ireland). Hydrolysates were tested at the concentration of 1 mg mL
1 in duplicate or triplicate. Test and control samples (25 mL per well) were directly added to cells using a Flex station II and fluorescent readings were taken for 80 s in flex mode with excitation and emission wavelengths of 485 and 525 nm, respectively. The change in intracellular Ca2þ was monitored as an increase in relative fluorescence units (RFU), and the difference between maximum and minimum RFU (VmaxeVmin) was compared and depicted as percentage of maximum Ca2þ influx as elicited by the controls (5-HT or FBS, as indicated). For dose response curves, data analysis was performed using PRISM 4.0 (GraphPAD Software Inc., San Diego, CA, USA). 2.5. Reverse-phase high performance liquid chromatography to quantify L-tryptophan and serotonin For quantification of L-tryptophan, the HPLC system consisted of a 510 pump, a 717 plus autosampler cooled at 4  C, a 486 absorbance detector, a bus SAT/IN module (Waters), a croco-cil column oven and a 1046A Fluorescent Detector (Hewlett Packard, supplied by Agilent Technologies, Dublin). All samples were injected onto a reversed-phase (RP) Luna 3 mm C18 150  2 mm column

58

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

(Phenomenex, London, UK), mounted with a Krudkatcher precolumn filter and security guard cartridges (Phenomenex). The analytical method was adapted from Herve, Beyne, Jamault, and Delacoux (1996). The mobile phase consisted of 50 mM acetic acid (Alkem/Reagecon, Cork, Ireland), 100 mM zinc acetate (Alkem/ Reagecon) with 3% (v/v) acetonitrile (Alkem/Reagecon) and was filtered through a 0.45 mm Millipore Durapore filter (AGB, Dublin) and vacuum degassed prior to use. Separations were achieved by isocratic elution at 0.3 mL min
1. The fluorescent detector was set to excitation and emission wavelengths of 254 and 404 nm, respectively. The UV detector was set at 330 nm. Samples were deproteinised by the addition of 20 mL of 4 M perchloric acid (Alkem/Reagecon) to 200 mL of sample spiked with 3-nitro-L-tyrosine (SigmaeAldrich) as internal standard. A 20 mL aliquot of sample or standard (L-tryptophan, SigmaeAldrich) was injected onto the HPLC column and results were processed using EmpowerÒ software (Waters). Analytes were identified based on their retention time and concentrations were determined using analyte:internal standard peak height ratios that were compared with standard injections. Results were expressed as ng Ltryptophan mg
1 of sample. Serotonin concentration was determined using a different RPHPLC system according to a modification of a previously described procedure (O’Mahony et al., 2008). The HPLC system consisted of a SCL 10-Avp system controller, LC-10AS pump, SIL-10A autoinjector (with sample cooler maintained at 4  C), CTO-10A oven, LECD 6A electrochemical detector (Shimadzu, Kyoto, Japan) and an online Gastorr degasser (ISS, Kent, UK). A RP column Kinetex 2.6 mm C18 100  4.6 mm (Phenomenex) maintained at 30  C was employed for analyte separation at a flow rate 0.9 mL min
1. The glassy carbon working electrode combined with an Ag/AgCl reference electrode (Shimadzu) was operated a þ0.8 V and the results were analysed using Class-VP 5 software (Shimadzu). The mobile phase, which contained 0.1 M citric acid, 0.1 M sodium dihydrogen phosphate, 0.01 mM EDTA (Alkem/Reagecon), 5.6 mM octan-1sulphonic acid (SigmaeAldrich) and 9% (v/v) methanol (Alkem/ Reagecon), was adjusted to pH 2.8 using 4 M sodium hydroxide (Alkem/Reagecon). Briefly, samples were diluted either 1/3 or 1/10 in chilled (4  C) mobile phase spiked with 0.1 ng mL
1 of N-methyl 5-HT (SigmaeAldrich) as internal standard. Diluted samples were then centrifuged at 19,098  g (Hettich MIKRO 22 R, DJB Labcare, UK) for 15 min at 4  C and 20 mL of the supernatant was injected onto the HPLC column. Serotonin was identified by its retention time as determined from injection of serotonin standards. Serotonin concentrations were determined using the analyte:internal standard (N-methyl 5-HT) peak height ratios, which were compared with that of serotonin standard injections. Results were expressed as ng serotonin mg
1 sample. 2.6. Cumulative food intake experiment Animals, male C57Bl/6 mice were purchased from Harlan (Blackthorn, UK). Mice aged 7e8 weeks were received at the facility. Animals were group-housed (4e5 mice per cage) in standard holding cages with controlled lightedark cycle (12 h light; lights on at 7.45 a.m.) and in a temperature- (21  1  C) and humiditycontrolled (55  10%) environment. Water and standard lab chow (2018S Teklad Global 18% Protein Rodent Diet, Harlan, UK) were available ad libitum. All mice were weighed (precision 0.1 g) twice weekly and on the day of the study between 9.00 and 10.00 a.m. All experiments were in full accordance with the European Community Council directive (86/609/EEC) and approved by the Animal Experimentation Ethics Committee of University College Cork. Cumulative food intake was analysed during the light cycle following a 16 h fast during the dark phase as described previously

(Vickers, Dourish, & Kennett, 2001). The fasting period prior to the study is warranted to induce a robust baseline feeding response, enabling studies with fewer animals. On the day of the study, mice were placed into individual housing cages and left to acclimatise for >15 min. Animals were randomised into two groups (n ¼ 10) and received an intraperitoneal (IP) injection with either vehicle (HBSS and 20 nM HEPES) or milk protein hydrolysate (WPH-1 kDa permeate or NaCNH-1 kDa permeate) resuspended in HBSS and 20 nM HEPES. The milk protein hydrolysates were administered at 200 or 500 mg kg
1 body weight and animals were returned to their individual holding cages. A pre-weighed amount (5.00  0.01 g) of standard lab chow was placed in the cages 20 min after IP injection of the sample or vehicle. Food intake was determined at regular intervals by measuring the cumulative amount of food consumed. 2.7. Statistical analysis Statistical analyses were performed using SPSS software (IBM SPSS statistics 20, Chicago, IL, USA). A mean multi comparison for the Ca2þ mobilisation assay was performed using one way analysis of variance (ANOVA) followed by Bonferroni post-hoc tests at a significance level of p < 0.05. Food intake measurements between groups were analysed using a one way, repeated measures ANOVA followed by estimation of the parameters. If the data was nonspherical a HuynheFeldt correction was applied. Graphs were expressed as mean  SEM. 3. Results and discussion 3.1. Serotonergic activity of WPH and its associated ultrafiltration fractions In this study, WP-derived enzymatic hydrolysates were exposed to Hek cells expressing two isoforms of the 5-HT2C receptor, the unedited 5-HT2C-INI receptor or the partially edited 5-HT2C-VSV receptor isoform. The partly edited 5-HT2C-VSV results from the distinctive ability of the 5-HT2C receptor to be modified by posttranscriptional RNA editing on 5 specific nucleotide positions, converting an adenosine to inosine residues, causing amino acid sequence changes (Burns et al., 1997). It is the most abundantly expressed 5-HT2C receptor isoform in human brain regions, in particularly in the hypothalamus (Niswender, Copeland, HerrickDavis, Emeson, & Sanders-Bush, 1999; Werry, Loiacono, Sexton, & Christopoulos, 2008). In addition, increased editing of the 5-HT2C receptor has been associated with an altered feeding behaviour and fat mass, supporting the role for the 5-HT2C receptor in obesity (Kawahara et al., 2008; Olaghere da Silva et al., 2010; Schellekens et al., 2012). An in-house established medium throughput cellular based screening platform was utilised to screen whey proteinderived hydrolysates for their 5-HT2C receptor activating potential. The WPHs sampled at different hydrolysis times were able to significantly induce intracellular Ca2þ mobilisation in Hek cells stably expressing both isoforms of the 5-HT2C receptor compared with wild type cells, albeit lower in cells expressing the partially edited 5-HT2C-VSV isoform (Fig. 2A). This was expected, as increased 5-HT2C receptor editing has been associated with a decreased receptor signalling (Berg, Clarke, Cunningham, & Spampinato, 2008; Burns et al., 1997; Niswender et al., 1999; Olaghere da Silva et al., 2010; Schellekens et al., 2012). Interestingly, unhydrolysed WP was also able to increase intracellular Ca2þ to some extent in cells expressing the 5-HT2C receptor isoforms. The relative Ca2þ influx in Hek cells expressing the 5-HT2C-INI isoform was similar at the different hydrolysis times studied. However, with the 5-HT2C-VSV isoform, an increase in relative Ca2þ influx was

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

59

Table 1 Increased whey protein hydrolysate (WPH)-mediated intracellular calcium (Ca2þ) in Hek-5-HT2C-INI cells upon enrichment of low molecular mass peptides in the ultrafiltration samples. Samplea

Relative increase in intracellular Ca2þ (% RFU)b

WP WPH WPH-10 kDa Ret WPH-10 kDa Perm WPH-5 kDa Ret WPH-5 kDa Perm WPH-1 kDa Ret WPH-1 kDa Perm


11.8 40.7
0.5 67.9 42.8 86.7 76.7 97.1

       

0.9 2.2a 2.6b 2.7a,b 3.2a,b 3.5a,b 1.3a,b 1.8a,b

a Abbreviations are: WP, whey protein; WPH, 240 min whey protein hydrolysate; Perm, ultrafiltration (UF) permeate; Ret, UF retentate. b Relative increased in intracellular Ca2þ expressed as percentage of relative fluorescence units (RFU) with respect to serotonin (10 nM); samples were tested at 0.5 mg mL
1. Values are mean  SEM of three replicates; superscripts a and b indicate statistically different values compared with WP and WPH, respectively (p < 0.001).

Fig. 2. Panel A: Whey protein hydrolysate (WPH)-mediated intracellular calcium (Ca2þ) in human embryonic kidney (Hek) wild type (wt) (,) cells, Hek-5-HT2C-INI ( ) and Hek-5-HT2C-VSV (-) cells. Intracellular Ca2þ increase was depicted as a percentage of maximal Ca2þ increase as elicited by the control (3.3% FBS). Graph represents the mean  SEM of triplicate samples analysed in duplicate. Statistically different values are indicated for Ca2þ increases in Hek-5-HT2C-INI cells compared with WP in Hek wt (a, p < 0.001); for Hek-5-HT2C-VSV compared with WP in Hek wt (b, p < 0.01); for Hek5-HT2C-INI compared with WP in Hek-5-HT2C-INI (c, p < 0.01 and d, p < 0.001) and for Hek-5-HT2C-VSV compared with WP in Hek-5-HT2C-VSV (e, p < 0.001). Panel B: WPH-1 kDa permeate (,) and the NaCNH-1 kDa permeate (C) induction of Ca2þ mobilisation in cells expressing the 5-HT2C receptor as analysed in a concentration response curve. Intracellular Ca2þ increase was depicted as a percentage of maximal Ca2þ increase as elicited by the endogenous ligand, 5-HT (1 mM). Graph represents the mean  SEM of duplicate samples (B).

observed with increased hydrolysis time. In a previous study it was also demonstrated that with NaCNH, the 5-HT2C receptor mediated Ca2þ influx was significantly (p < 0.05) enhanced with increasing hydrolysis time (Nongonierma et al., 2013). Indeed, NaCNH was shown to induce a Ca2þ influx >60% of 5-HT2C receptor mediated Ca2þ signalling compared with control. In contrast, we demonstrate that WPHs herein were only able to mediate 50% of 5-HT2C receptor mediated Ca2þ signalling compared with the control (Fig. 2A). Nevertheless, the independent duplicate of WPH gave similar activation of the serotonin 5-HT2C receptor (Table 1 and Fig. 2A), suggesting that generation of the bioactive peptides was reproducible. Next, UF fractionation of the WPH 240 min hydrolysate was carried out. The RP-UPLC profiles of WP, WPH and its associated UF fractions are illustrated in Fig. 3. The WPH (Fig. 3B) still contained unhydrolysed whey proteins (Fig. 3A), notably a high proportion of b-lactoglobulin was still present within the hydrolysate. This is in agreement with the relatively high resistance of b-lactoglobulin to peptic hydrolysis (Reddy, Kella, & Kinsella, 1988). In agreement with

the cut-off of the membranes, the unhydrolysed proteins were retained in the 10 kDa retentate (Fig. 3C) and the 10, 5 and 1 kDa permeates did not contain any unhydrolysed WPs (Fig. 3D, E and 3F). The relative 5-HT2C receptor-mediated increase in intracellular Ca2þ was significantly higher in the 1, 5 and 10 kDa permeates compared with their associated retentates (Table 1). A one-way ANOVA comparisons revealed an overall significant increase in 5HT2C receptor activation with UF fractionation (Table 1) of F(7) ¼ 270.120; p < 0.001. The molecular mass distribution profile of WPH and its associated UF fractions is illustrated in Fig. 4. An enrichment in low molecular mass peptides was seen during sequential UF fractionation, with the 1 kDa permeate containing w92% peptides <1 kDa. The 5-HT2C receptor-mediated calcium increases were significantly higher in UF fractions enriched with low molecular mass peptides compared with unhydrolysed WP and WPH 240 min (p < 0.001) (Table 1). This indicates that the low molecular mass peptides within the UF permeates were responsible for activation of the serotonin 5-HT2C receptor. Similar results were previously found with the NaCNH, also showing an increase in the 5-HT2C receptor activation with peptides <1 kDa (Nongonierma et al., 2013). Comparison of concentration response curves for NaCNH-1 kDa and WPH-1 kDa permeates demonstrated a similar efficacy (75e 100%) (Fig. 2B). However, the WPH-1 kDa permeate had a significantly higher (p < 0.05) potency when compared with NaCNH1 kDa permeate (Fig. 2B), with absolute half maximal effective concentration (EC50) values of 0.022 and 0.111 mg mL
1, respectively. 3.2. Specificity of the NaCNH and WPH 1 kDa permeate samples across the serotonin 2 receptor family As already outlined, the 5-HT2 receptor family constitutes three different receptor types, the 5-HT2A, 5-HT2B and the 5-HT2C receptors (Hoyer et al., 1994; Hoyer, Hannon, & Martin, 2002). Activation of these receptors has been implicated in several physiological functions of the CNS and in pathologies including anxiety, depression, migraine, satiety and schizophrenia (Baxter, Kennett, Blaney, & Blackburn, 1995). Previous studies have identified that activation of the 5-HT2B receptor by serotonergic drugs, including Fenfluramine, plays an important role in the occurrence of negative side-effects, including pulmonary hypertension, impaired regulation of plasma serotonin level and valvulopathy (Miller, 2005; Rothman et al., 2000).

60

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

Fig. 3. Reverse-phase ultra-performance liquid chromatographic (RP-UPLC) profile of (A) the unhydrolysed whey protein (WP), (B) 240 min whey protein hydrolysate (WPH), (C) 10 kDa retentate of WPH, (D) 10 kDa permeate of WPH (E) 5 kDa permeate of WPH and (F) the 1 kDa permeate of WPH. 1: caseinomacropeptide; 2: a-lactalbumin; 3: b-lactoglobulin. The RP-UPLC profiles depicted are from a single experiment. The analysis was conducted three times for the 240 min hydrolysate (n ¼ 3), showing identical peptide profiles in the 3 instances.

The first specific 5-HT2C receptor agonist, Lorcaserin, was recently approved for the treatment of obesity (Jandacek, 2005; Martin et al., 2011; O’Neil et al., 2012; Redman & Ravussin, 2010), reinforcing the importance of the 5-HT2C receptor as a target for weight management. Nevertheless, serotonergic drugs, including Lorcaserin, are associated with safety concerns due to non-

specificity, in particular caused by the non-selective activation of the 5-HT2A and 5-HT2B receptors at the therapeutic dose. This has been suggested to be causal to heart and pulmonary vasculature side-effects (Miller, 2005). The development of natural satiety inducing bioactives, such as milk protein-derived bioactive peptides specifically targeting the 5HT2C receptor, may help to avoid the side-effects generally associated with the utilisation of pharmaceutical drugs. We therefore set out to investigate the 5-HT2C receptor specificity for the most active compounds, i.e., the NaCNH-1 kDa and WPH-1 kDa permeates (Fig. 5). Both 1 kDa permeates induced significant Ca2þ mobilisation in cells expressing the 5-HT2C receptor but not in cells expressing either the 5-HT2A or 5-HT2B receptor, while 5-HT was shown to equally activate the three 5-HT2 receptors when tested at 1 mg mL
1 (Fig. 5). This shows that the milk-derived 1-kDa permeates are more specific for the 5-HT2C receptor compared with the 5-HT2A and 5-HT2B receptor, which indicates an advantageous safety profile. 3.3. Serotonin and L-tryptophan concentrations in the NaCNH and WPH 1 kDa permeates

Fig. 4. Molecular mass distribution (-, >5 kDa; ,, <1 kDa, , 5-1 kDa) of the 240 min whey protein hydrolysate (WPH) and its associated ultrafiltration (UF) fractions.

It is well known that 5-HT and the 5-HT precursor L-tryptophan have appetite-suppressing activities. Therefore, 5-HT and free Ltryptophan concentrations within WP, WPH 240 min and the WPH1 kDa permeate were determined. In addition, 5-HT and free Ltryptophan concentrations were determined in NaCN, NaCNH 240 min and the NaCNH 1 kDa permeate. None of the samples

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

Fig. 5. Specificity of the NaCNH-1 kDa permeate and WPH-1 kDa permeate samples compared with 5-HT2 receptor activation mediated by the endogenous ligand, 5-HT (1 mM). Intracellular calcium (Ca2þ) increase was depicted as a percentage of maximal Ca2þ increase as elicited by the control (1 mM 5HT) in Hek-5-HT2A (,) cells, Hek-5-HT2B ( ) and Hek-5-HT2C (-) cells. Graph represents the mean  SEM of triplicate samples for 5HT and duplicate samples for the NaCNH-1 kDa and WPH-1 kDa samples, respectively. Statistically different values of 5-HT2C compared with 5-HT2A (a; p < 0.001 and c; p < 0.01) and compared with 5-HT2B (b; p < 0.001) are indicated.

contained 5-HT. Moreover, unhydrolysed WP and NaCN were also void of free L-tryptophan. The NaCNH 240 min and WPH 240 min samples contained very low concentrations of free L-tryptophan, i.e., 6 and 11 ng mg
1, respectively. The WPH and NaCNH-1 kDa permeates contained high to moderate concentrations of L-tryptophan, i.e., 114 and 25 ng mg
1, respectively. Although L-tryptophan cannot directly bind the 5-HT2C receptor, it may contribute to potential satiety effects overall in vivo. However, this cannot explain the potent direct activation of the 5-HT2C receptor in vitro by the WPH-1 kDa permeate. This suggests that milk protein-derived peptides within NaCNH-1 kDa permeate were responsible for activation of the 5-HT2C receptor. 3.4. Effect of NaCNH and WPH 1 kDa permeates on cumulative food intake in mice Milk protein-derived bioactives have been shown to stimulate the release of various gut hormones associated with the regulation of food intake, including cholecystokinin (CCK) (Bowen, Noakes, Trenerry, & Clifton, 2006b; Figlewicz et al., 1992; Hall, Millward, Long, & Morgan, 2003), peptide YY (PYY) (Calbet & Holst, 2004; le Roux & Bloom, 2005), glucagon-like peptide 1 (GLP-1) (Aziz & Anderson, 2003; Hall et al., 2003) and ghrelin (Bowen, Noakes, & Clifton, 2006a; Bowen et al., 2006b). Lactoferrin has recently been reported to reduce abdominal obesity via modulation of lipopolysaccharide levels and composition (Ono, Morishita, & Murakoshi, 2013). In humans, chronic intake of lactoferrin has also been associated with a decrease in visceral fat (Ono et al., 2010). Similarly, intake of a-lactalbumin has been reported to reduce hunger, increase energy expenditure, and to regulate protein and fat balance (Hursel, van der Zee, & Westerterp-Plantenga, 2010). The satiety effects of a-lactalbumin have been linked with the higher bioavailability of tryptophan, the 5-HT precursor. Thus, ingestion of a-lactalbumin increases 5-HT release in rats (Orosco et al., 2004) and in humans (Booij et al., 2006; Nieuwenhuizen et al., 2009), as a consequence. The effect of the NaCNH and WPH 1 kDa permeates on cumulative food intake in male mice (C57Bl/6) was studied. No effect on food intake was seen following intraperitoneal administration of 200 mg kg
1 body weight (Fig. 6A and B). Nevertheless, following

61

500 mg kg
1 body weight administration of the NaCNH-1 kDa permeate, a significant reduction of cumulative food intake was observed (Fig. 6C and D). Repeated measures ANOVA showed a significant main effect of the treatment NaCNH-1 kDa permeate versus vehicle [F(1,18) ¼ 8.552; p < 0.01] and a significant main effect of time [F(4.447,80.049) ¼ 834.962; p < 0.001]. However, no significant interaction of time  treatment [F(4.447,80.049) ¼ 1.429; p ¼ 0.228] was seen. Unhydrolysed WP and NaCN did not demonstrate a significant main effect on food intake when compared with vehicle, confirming the specificity of the anorexigenic effect of the NaCNH-1 kDa permeate (Fig. 6G and H). Surprisingly, no significant effect on food intake was observed following IP administration of the WPH-1 kDa permeate when tested at 500 mg kg
1 body weight (Fig. 6E and F). The lack of bioactivity seen with WPH-1 kDa permeate may be explained by the fact that the bioactive peptides within this sample may not be bioavailable possibly due to their degradation by peptidases in the circulation or to a poor uptake in the target tissues. Future studies are needed to assess the an orexigenic effect of these dairy-derived peptides following oral ingestion as well as the resulting plasma concentrations and kinetics of these orally administered bioactives. Bioactive peptides within the milk protein hydrolysates investigated in this study need to be able to cross the gut barrier and resist degradation by epithelial and serum peptidases. Previously, it has been shown that only minor fractions of dietary peptides reach the blood circulation due to proteolytic instability and poor absorption. However, intestinal transport of intact peptides has been reported in vivo (Gardner, 1983). The bioavailability of peptides is thought to be governed by their stability to gastrointestinal digestion, their permeability through the intestinal mucosa and stability to brush border and serum peptidases. Casein-derived bioactive peptides could be detected in the jejunum of human subjects intubated with a nasogastric tube (Boutrou et al., 2013). Transepithelial transport of oligopeptides through a Caco-2 cell layer has been reported to be mainly governed by the hydrolytic activity of brush border peptidases (Shimizu, Tsunogai, & Arai, 1997). Degradation of the angiotensin converting enzyme (ACE) inhibitory peptide, LeueHiseLeueProe LeuePro, by brush border peptidases of Caco-2 cells has been reported to occur as early as 5 min (Quirós, Dávalos, Lasunción, Ramos, & Recio, 2008). However, the peptide (HiseLeueProe LeuePro) released by brush border peptidases was still bioactive and could cross the Caco-2 cell layer. Degradation of ValeProePro has also been shown, but intact material could be found in the basolateral compartment, showing that a certain proportion of ValeProePro could cross the Caco-2 cell monolayer without being degraded (Satake et al., 2002). A few studies have demonstrated that specific, sequences could reach the blood circulation intact without gastrointestinal degradation. Following ingestion of a lacto-tripeptide enriched yogurt beverage, the ACE inhibitory peptide, IleeProePro was found in its intact format in the blood circulation (Foltz et al., 2007). Other relatively hydrophobic dipeptides (LeueTrp, Phee Tyr and IleeTyr) were identified in the plasma following ingestion of the lacto-tripeptide enriched yoghurt beverage (Foltz et al., 2007). Moreover, the bioactive peptides found within hydrolysed NaCN are relatively small and hydrophobic in nature, which may suggest that they have the potential to survive gastrointestinal digestion and allow for intestinal permeation (Morifuji et al., 2010; Nongonierma et al., 2013; Shimizu et al., 1997). Larger peptides may also cross the intestinal barrier and it has been reported that peptides 5 amino acids could permeate through a layer of Caco-2 cells (Quirós et al., 2008; Vermeirssen et al., 2002).

62

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

Fig. 6. Cumulative food intake following administration of milk protein-derived hydrolysates. Food (regular chow) intake in C57Bl/6 male mice was determined following intraperitoneal (IP) injection with 200 mg kg
1 body weight (A, B) or 500 mg kg
1 body weight (C, D) 1 kDa permeate of the 240 min sodium caseinate hydrolysate (NaCNH-1 kDa permeate). The 1 kDa permeate of the 240 min whey protein hydrolysate (WPH-1 kDa permeate) was IP injected at 500 mg kg
1 body weight (E, F). For panels AeF: C, vehicle; ,, hydrolysate. Unhydrolysed WP and NaCN were also IP injected at 500 mg kg
1 body weight (G, H; C, vehicle; ,, unhydrolysed WP; :, unhydrolysed NaCN). Cumulative food intake was determined at regular intervals after IP injection (A, C, E and G). Food intake per time bin was calculated (B, D, F and H). Graphs represents the mean  SEM of n ¼ 9 (G, H) or 10 (AeF) mice per treatment group. Statistical significance was determined using repeated measures ANOVA and estimation of parameters; statistical significance depicted is notated as **p < 0.01 and *p < 0.05.

4. Conclusion In this study the 5-HT2C receptor activating potential of enzymatic hydrolysates of WP and NaCN was demonstrated in vitro. It was shown that the serotonergic activation observed with the

WHP-1 kDa permeate and the NaCNH-1 kDa permeate was specific for the 5-HT2C receptor, with no activation seen for the 5-HT2A and 5-HT2B receptors. This suggests that the bioactive peptides may induce fewer side effects generally associated with non-specific activation of the 5-HT2B receptor, which may lead to

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

valvulopathy. The in vitro 5-HT2C activating potential of NaCNH-1 kDa permeate translated to a reduced cumulative food intake in mice. However, these effects were only observed at a high dose of sample following IP administration. In addition, no in vivo effect was seen upon administration of the WHP-1 kDa permeate, despite the high potency on 5-HT2C receptor activation in vitro. The reason for the absence of an in vivo effect of the WPH derived hydrolysate permeate could be multifaceted and may be related to peptide stability, bioavailability or mal-absorption, and should be investigated further. The results described herein demonstrate the potential of milk-protein derived bioactive peptides with 5-HT2C receptor agonist properties as satiating ingredients. These may be further exploited in the development of functional foods with antiobesity properties.

Acknowledgement The work described herein was supported by Enterprise Ireland under Grant Number TC2013-0001.

References Anderson, G. H., Tecimer, S. N., Shah, D., & Zafar, T. A. (2004). Protein source, quantity, and time of consumption determine the effect of proteins on shortterm food intake in young men. Journal of Nutrition, 134, 3011e3015. Aronne, L. J., Powell, A. G., & Apovian, C. M. (2011). Emerging pharmacotherapy for obesity. Expert Opinion on Emerging Drugs, 16, 587e596. Aziz, A., & Anderson, G. H. (2003). Exendin-4, a GLP-1 receptor agonist, interacts with proteins and their products of digestion to suppress food intake in rats. Journal of Nutrition, 133, 2326e2330. Baxter, G., Kennett, G., Blaney, F., & Blackburn, T. (1995). 5-HT2 receptor subtypes: a family re-united? Trends in Pharmacological Sciences, 16, 105e110. Berg, K. A., Clarke, W. P., Cunningham, K. A., & Spampinato, U. (2008). Fine-tuning serotonin2c receptor function in the brain: molecular and functional implications. Neuropharmacology, 55, 969e976. Bloom, S. R., Kuhajda, F. P., Laher, I., Pi-Sunyer, X., Ronnett, G. V., Tan, T. M., et al. (2008). The obesity epidemic: pharmacological challenges. Molecular Interventions, 8, 82e98. Booij, L., Merens, W., Markus, C. R., & Van der Does, A. J. (2006). Diet rich in alphalactalbumin improves memory in unmedicated recovered depressed patients and matched controls. Journal of Psychopharmacology, 20, 526e535. Boutrou, R., Gaudichon, C., Dupont, D., Jardin, J., Airinei, G., Marsset-Baglieri, A., et al. (2013). Sequential release of milk protein-derived bioactive peptides in the jejunum in healthy humans. American Journal of Clinical Nutrition, 97, 1314e 1323. Bowen, J., Noakes, M., & Clifton, P. M. (2006a). Appetite regulatory hormone responses to various dietary proteins differ by body mass index status despite similar reductions in ad libitum energy intake. Journal of Clinical Endocrinology and Metabolism, 91, 2913e2919. Bowen, J., Noakes, M., Trenerry, C., & Clifton, P. M. (2006b). Energy intake, ghrelin, and cholecystokinin after different carbohydrate and protein preloads in overweight men. Journal of Clinical Endocrinology and Metabolism, 91, 1477e 1483. Burns, C. M., Chu, H., Rueter, S. M., Hutchinson, L. K., Canton, H., Sanders-Bush, E., et al. (1997). Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature, 387, 303e308. Burton-Freeman, B. M. (2008). Glycomacropeptide (GMP) is not critical to wheyinduced satiety, but may have a unique role in energy intake regulation through cholecystokinin (CCK). Physiology and Behavior, 93, 379e387. Calbet, J. A., & Holst, J. J. (2004). Gastric emptying, gastric secretion and enterogastrone response after administration of milk proteins or their peptide hydrolysates in humans. European Journal of Clinical Nutrition, 43, 127e139. Cecchini, M., Sassi, F., Lauer, J. A., Lee, Y. Y., Guajardo-Barron, V., & Chisholm, D. (2010). Tackling of unhealthy diets, physical inactivity, and obesity: health effects and cost-effectiveness. Lancet, 376, 1775e1784. Chakrabarti, R. (2009). Pharmacotherapy of obesity: emerging drugs and targets. Expert Opinion on Therapeutic Targets, 13, 195e207. Dalton, G. L., Lee, M. D., Kennett, G. A., Dourish, C. T., & Clifton, P. G. (2004). mCPP-induced hyperactivity in 5-HT2C receptor mutant mice is mediated by activation of multiple 5-HT receptor subtypes. Neuropharmacology, 46, 663e671. Dalton, G. L., Lee, M. D., Kennett, G. A., Dourish, C. T., & Clifton, P. G. (2006). Serotonin 1B and 2C receptor interactions in the modulation of feeding behaviour in the mouse. Psychopharmacology (Berlin), 185, 45e57. Derosa, G., & Maffioli, P. (2012). Anti-obesity drugs: a review about their effects and their safety. Expert Opinion on Drug Safety, 11, 459e471.

63

Dougkas, A., Reynolds, C. K., Givens, I. D., Elwood, P. C., & Minihane, A. M. (2011). Associations between dairy consumption and body weight: a review of the evidence and underlying mechanisms. Nutrition Research Reviews, 1e24. Dutton, A. C., & Barnes, N. M. (2006). Anti-obesity pharmacotherapy: future perspectives utilising 5-HT2C receptor agonists. Drug Discovery Today: Therapeutic Strategies, 3, 577e583. Figlewicz, D. P., Nadzan, A. M., Sipols, A. J., Green, P. K., Liddle, R. A., Porte, D., Jr., et al. (1992). Intraventricular CCK-8 reduces single meal size in the baboon by interaction with type-A CCK receptors. American Journal of Physiology, 263, R863eR867. FitzGerald, R. J., Murray, B. A., & Walsh, D. J. (2004). Hypotensive peptides from milk proteins. Journal of Nutrition, 134, 980Se988S. Follenzi, A., & Naldini, L. (2002a). Generation of HIV-1 derived lentiviral vectors. Methods in Enzymology, 346, 454e465. Follenzi, A., & Naldini, L. (2002b). HIV-based vectors. Preparation and use. Methods in Molecular Medicine, 69, 259e274. Foltz, M., Meynen, E. E., Bianco, V., van Platerink, C., Koning, T. M., & Kloek, J. (2007). Angiotensin converting enzyme inhibitory peptides from a lactotripeptideenriched milk beverage are absorbed intact into the circulation. Journal of Nutrition, 137, 953e958. Froetschel, M. A., Azain, M. J., Edwards, G. L., Barb, C. R., & Amos, H. E. (2001). Opioid and cholecystokinin antagonists alleviate gastric inhibition of food intake by premeal loads of casein in meal-fed rats. Journal of Nutrition, 131, 3270e3276. Gardner, M. L. G. (1983). Entry of peptides of dietary origin into the circulation. Nutrition and Health, 2, 163e171. Garfield, A. S., & Heisler, L. K. (2009). Pharmacological targeting of the serotonergic system for the treatment of obesity. Journal of Physiology, 587, 49e60. Gonzalez-Castejon, M., & Rodriguez-Casado, A. (2011). Dietary phytochemicals and their potential effects on obesity: a review. Pharmacological Research, 64, 438e455. Gooda Sahib, N., Saari, N., Ismail, A., Khatib, A., Mahomoodally, F., & Abdul Hamid, A. (2012). Plants metabolites as potential antiobesity agents. Scientific World Journal, 2012, 8. Halford, J. C., Boyland, E. J., Blundell, J. E., Kirkham, T. C., & Harrold, J. A. (2010). Pharmacological management of appetite expression in obesity. Nature Reviews Endocrinology, 6, 255e269. Halford, J. C., Harrold, J. A., Boyland, E. J., Lawton, C. L., & Blundell, J. E. (2007). Serotonergic drugs: effects on appetite expression and use for the treatment of obesity. Drugs, 67, 27e55. Hall, W. L., Millward, D. J., Long, S. J., & Morgan, L. M. (2003). Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite. British Journal of Nutrition, 89, 239e248. Herve, C., Beyne, P., Jamault, H., & Delacoux, E. (1996). Determination of tryptophan and its kynurenine pathway metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet and fluorimetric detection. Journal of Chromatography B: Biomedical Sciences and Applications, 675, 157e161. Hoyer, D., Clarke, D. E., Fozard, J. R., Hartig, P. R., Martin, G. R., Mylecharane, E. J., et al. (1994). International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacological Reviews, 46, 157e203. Hoyer, D., Hannon, J. P., & Martin, G. R. (2002). Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacology Biochemistry and Behavior, 71, 533e554. Hursel, R., van der Zee, L., & Westerterp-Plantenga, M. S. (2010). Effects of a breakfast yoghurt, with additional total whey protein or caseinomacropeptidedepleted alpha-lactalbumin-enriched whey protein, on diet-induced thermogenesis and appetite suppression. British Journal of Nutrition, 103, 775e780. Jandacek, R. J. (2005). APD-356 (arena). Current Opinion in Investigational Drugs, 6, 1051e1056. Kang, J. G., & Park, C. Y. (2012). Anti-obesity drugs: a review about their effects and safety. Journal of Diabetes and Metabolism, 36, 13e25. Kawahara, Y., Grimberg, A., Teegarden, S., Mombereau, C., Liu, S., Bale, T. L., et al. (2008). Dysregulated editing of serotonin 2C receptor mRNAs results in energy dissipation and loss of fat mass. Journal of Neuroscience, 28, 12834e12844. Kennett, G. A., & Clifton, P. G. (2010). New approaches to the pharmacological treatment of obesity: can they break through the efficacy barrier? Pharmacology Biochemistry and Behavior, 97, 63e83. Korhonen, H. (2009). Milk-derived bioactive peptides: from science to applications. Journal of Functional Foods, 1, 177e187. Korhonen, H., Marnila, P., & Gill, H. S. (2000). Bovine milk antibodies for health. British Journal of Nutrition, 84, S135eS146. Kreider, R. B., Iosia, M., Cooke, M., Hudson, G., Rasmussen, C., Chen, H., et al. (2011). Bioactive properties and clinical safety of a novel milk protein peptide. Nutrition Journal, 10, 99. Kumar, M., Verma, V., Nagpal, R., Kumar, A., Behare, P. V., Singh, B., et al. (2012). Anticarcinogenic effect of probiotic fermented milk and chlorophyllin on aflatoxin-B1-induced liver carcinogenesis in rats. British Journal of Nutrition, 107, 1006e1016. Lam, D. D., Przydzial, M. J., Ridley, S. H., Yeo, G. S., Rochford, J. J., O’Rahilly, S., et al. (2008). Serotonin 5-HT2C receptor agonist promotes hypophagia via downstream activation of melanocortin 4 receptors. Endocrinology, 149, 1323e1328. Lonnerdal, B. (2003). Nutritional and physiologic significance of human milk proteins. American Journal of Clinical Nutrition, 77, 1537Se1543S. Luhovyy, B. L., Akhavan, T., & Anderson, G. H. (2007). Whey proteins in the regulation of food intake and satiety. Journal of the American College of Nutrition, 26, 704Se712S.

64

H. Schellekens et al. / International Dairy Journal 38 (2014) 55e64

Martin, C. K., Redman, L. M., Zhang, J., Sanchez, M., Anderson, C. M., Smith, S. R., et al. (2011). Lorcaserin, a 5-HT(2C) receptor agonist, reduces body weight by decreasing energy intake without influencing energy expenditure. Journal of Clinical Endocrinology and Metabolism, 96, 837e845. van Meijl, L. E., Vrolix, R., & Mensink, R. P. (2008). Dairy product consumption and the metabolic syndrome. Nutrition Research Reviews, 21, 148e157. Meisel, H., & FitzGerald, R. J. (2003). Biofunctional peptides from milk proteins: mineral binding and cytomodulatory effects. Current Pharmaceutical Design, 9, 1289e1295. Miller, K. J. (2005). Serotonin 5-ht2c receptor agonists: potential for the treatment of obesity. Molecular Interventions, 5, 282e291. Morifuji, M., Ishizaka, M., Baba, S., Fukuda, K., Matsumoto, H., Koga, J., et al. (2010). Comparison of different sources and degrees of hydrolysis of dietary protein: effect on plasma amino acids, dipeptides, and insulin responses in human subjects. Journal of Agricultural and Food Chemistry, 58, 8788e8797. Nagpal, R., Behare, P., Rana, R., Kumar, A., Kumar, M., Arora, S., et al. (2011). Bioactive peptides derived from milk proteins and their health beneficial potentials: an update. Food and Function, 2, 18e27. Naldini, L. (1998). Lentiviruses as gene transfer agents for delivery to non-dividing cells. Current Opinion in Biotechnology, 9, 457e463. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 272, 263e267. Nguyen, N., Champion, J. K., Ponce, J., Quebbemann, B., Patterson, E., Pham, B., et al. (2012). A review of unmet needs in obesity management. Obesity Surgery, 22, 956e966. Nieuwenhuizen, A. G., Hochstenbach-Waelen, A., Veldhorst, M. A., Westerterp, K. R., Engelen, M. P., Brummer, R. J., et al. (2009). Acute effects of breakfasts containing alpha-lactalbumin, or gelatin with or without added tryptophan, on hunger, ‘satiety’ hormones and amino acid profiles. British Journal of Nutrition, 101, 1859e1866. Niswender, C. M., Copeland, S. C., Herrick-Davis, K., Emeson, R. B., & Sanders-Bush, E. (1999). RNA editing of the human serotonin 5-hydroxytryptamine 2C receptor silences constitutive activity. Journal of Biological Chemistry, 274, 9472e9478. Nongonierma, A. B., & FitzGerald, R. J. (2012a). Biofunctional properties of caseinophosphopeptides in the oral cavity. Caries Research, 46, 234e267. Nongonierma, A. B., & FitzGerald, R. J. (2012b). Tryptophan-containing milk proteinderived dipeptides inhibit xanthine oxidase. Peptides, 37, 263e272. Nongonierma, A. B., & FitzGerald, R. J. (2013). Dipeptidyl peptidase IV inhibitory and antioxidative properties of milk-derived dipeptides and hydrolysates. Peptides, 39, 157e163. Nongonierma, A. B., Schellekens, H., Dinan, T. G., Cryan, J. F., & Fitzgerald, R. J. (2013). Milk protein hydrolysates activate 5-HT2C serotonin receptors: influence of the starting substrate and isolation of bioactive fractions. Food and Function, 4, 728e 737. O’Mahony, S., Chua, A. S., Quigley, E. M., Clarke, G., Shanahan, F., Keeling, P. W., et al. (2008). Evidence of an enhanced central 5HT response in irritable bowel syndrome and in the rat maternal separation model. Neurogastroenterology and Motility, 20, 680e688. O’Neil, P. M., Smith, S. R., Weissman, N. J., Fidler, M. C., Sanchez, M., Zhang, J., et al. (2012). Randomized placebo-controlled clinical trial of Lorcaserin for weight loss in type 2 diabetes mellitus: the BLOOM-DM study. Obesity, 20, 1426e1436. Obici, S. (2009). Minireview: molecular targets for obesity therapy in the brain. Endocrinology, 150, 2512e2517. Olaghere da Silva, U. B., Morabito, M. V., Canal, C. E., Airey, D. C., Emeson, R. B., & Sanders-Bush, E. (2010). Impact of RNA editing on functions of the serotonin 2C receptor in vivo. Frontiers in Neuroscience, 4, 26. Ono, T., Morishita, S., & Murakoshi, M. (2013). Novel function of bovine lactoferrin in lipid metabolism: visceral fat reduction by enteric-coated lactoferrin. PharmaNutrition, 1, 32e34. Ono, T., Murakoshi, M., Suzuki, N., Iida, N., Ohdera, M., Iigo, M., et al. (2010). Potent anti-obesity effect of enteric-coated lactoferrin: decrease in visceral fat accumulation in Japanese men and women with abdominal obesity after 8-week administration of enteric-coated lactoferrin tablets. British Journal of Nutrition, 104, 1688e1695. Orosco, M., Rouch, C., Beslot, F., Feurte, S., Regnault, A., & Dauge, V. (2004). Alphalactalbumin-enriched diets enhance serotonin release and induce anxiolytic and rewarding effects in the rat. Behavioural Brain Research, 148, 1e10. Phelan, M., & Kerins, D. (2011). The potential role of milk-derived peptides in cardiovascular disease. Food and Function, 2, 153e167. Pilvi, T. K., Korpela, R., Huttunen, M., Vapaatalo, H., & Mervaala, E. M. (2007). Highcalcium diet with whey protein attenuates body-weight gain in high-fat-fed C57Bl/6J mice. British Journal of Nutrition, 98, 900e907. Pilvi, T. K., Storvik, M., Louhelainen, M., Merasto, S., Korpela, R., & Mervaala, E. M. (2008). Effect of dietary calcium and dairy proteins on the adipose tissue gene expression profile in diet-induced obesity. Journal of Nutrigenetics and Nutrigenomics, 1, 240e251. Powell, A. G., Apovian, C. M., & Aronne, L. J. (2011). New drug targets for the treatment of obesity. Clinical Pharmacology and Therapeutics, 90, 40e51. Powell, T. M., & Khera, A. (2010). Therapeutic approaches to obesity. Current Treatment Options in Cardiovascular Medicine, 12, 381e395.

Power, O., Jakeman, P., & FitzGerald, R. J. (2013). Antioxidative peptides: enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides. Amino Acids, 44, 797e820. Quirós, A., Dávalos, A., Lasunción, M. A., Ramos, M., & Recio, I. (2008). Bioavailability of the antihypertensive peptide LHLPLP: transepithelial flux of HLPLP. International Dairy Journal, 18, 279e286. Reddy, I. M., Kella, N. K. D., & Kinsella, J. E. (1988). Structural and conformational basis of the resistance of beta-lactoglobulin to peptic and chymotryptic digestion. Journal of Agricultural and Food Chemistry, 36, 737e741. Redman, L. M., & Ravussin, E. (2010). Lorcaserin for the treatment of obesity. Drugs Today (Barcelona), 46, 901e910. Ricci-Cabello, I., Herrera, M. O., & Artacho, R. (2012). Possible role of milk-derived bioactive peptides in the treatment and prevention of metabolic syndrome. Nutrition Reviews, 70, 241e255. Rothman, R. B., Baumann, M. H., Savage, J. E., Rauser, L., McBride, A., Hufeisen, S. J., et al. (2000). Evidence for possible involvement of 5-HT(2B) receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications. Circulation, 102, 2836e2841. le Roux, C. W., & Bloom, S. R. (2005). Peptide YY, appetite and food intake. Proceedings of the Nutrition Society, 64, 213e216. Satake, M., Enjoh, M., Nakamura, Y., Takano, T., Kawamura, Y., Arai, S., et al. (2002). Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro, in human intestinal Caco-2 cell monolayers. Biosciences, Biotechnology and Biochemistry, 66, 378e384. Schellekens, H., Clarke, G., Jeffery, I. B., Dinan, T. G., & Cryan, J. F. (2012). Dynamic 5HT2C receptor editing in a mouse model of obesity. PLoS One, 7, e32266. Schellekens, H., Dinan, T. G., & Cryan, J. F. (2009). Lean mean fat reducing “ghrelin” machine: hypothalamic ghrelin and ghrelin receptors as therapeutic targets in obesity. Neuropharmacology, 58, 2e16. Schellekens, H., van Oeffelen, W. E., Dinan, T. G., & Cryan, J. F. (2013). Promiscuous dimerization of the growth hormone secretagogue receptor (GHS-R1a) attenuates ghrelin-mediated signaling. Journal of Biological Chemistry, 288, 181e191. Shimizu, M., Tsunogai, M., & Arai, S. (1997). Transepithelial transport of oligopeptides in the human intestinal cell, Caco-2. Peptides, 18, 681e687. Sokolov, O. Y., Pryanikova, N. A., Kost, N. V., Zolotarev, Y. A., Ryukert, E. N., & Zozulya, A. A. (2005). Reactions between beta-casomorphins-7 and 5-HT2serotonin receptors. Bulletin of Experimental Biology and Medicine, 140, 582e 584. Somerville, E. M., Horwood, J. M., Lee, M. D., Kennett, G. A., & Clifton, P. G. (2007). 5HT(2C) receptor activation inhibits appetitive and consummatory components of feeding and increases brain c-fos immunoreactivity in mice. European Journal of Neuroscience, 25, 3115e3124. Spellman, D., O’Cuinn, G., & FitzGerald, R. J. (2009). Bitterness in Bacillus proteinase hydrolysates of whey proteins. Food Chemistry, 114, 440e446. Staljanssens, D., Van Camp, J., Billiet, A., De Meyer, T., Al Shukor, N., De Vos, W. H., et al. (2012). Screening of soy and milk protein hydrolysates for their ability to activate the CCK1 receptor. Peptides, 34, 226e231. Tecott, L. H. (2007). Serotonin and the orchestration of energy balance. Cell Metabolism, 6, 352e361. Tecott, L. H., Sun, L. M., Akana, S. F., Strack, A. M., Lowenstein, D. H., Dallman, M. F., et al. (1995). Eating disorder and epilepsy in mice lacking 5-HT2c serotonin receptors. Nature, 374, 542e546. Valentino, M. A., Lin, J. E., & Waldman, S. A. (2010). Central and peripheral molecular targets for antiobesity pharmacotherapy. Clinical Pharmacology and Therapeutics, 87, 652e662. Veldhorst, M. A., Nieuwenhuizen, A. G., Hochstenbach-Waelen, A., van Vught, A. J., Westerterp, K. R., Engelen, M. P., et al. (2009). Dose-dependent satiating effect of whey relative to casein or soy. Physiology and Behavior, 96, 675e682. Vermeirssen, V., Deplancke, B., Tappenden, K. A., Van Camp, J., Gaskins, H. R., & Verstraete, W. (2002). Intestinal transport of the lactokinin Ala-Leu-Pro-MetHis-Ile-Arg through a Caco-2 Bbe monolayer. Journal of Peptide Science, 8, 95e 100. Vickers, S. P., Clifton, P. G., Dourish, C. T., & Tecott, L. H. (1999). Reduced satiating effect of d-fenfluramine in serotonin 5-HT(2C) receptor mutant mice. Psychopharmacology (Berlin), 143, 309e314. Vickers, S. P., Dourish, C. T., & Kennett, G. A. (2001). Evidence that hypophagia induced by d-fenfluramine and d-norfenfluramine in the rat is mediated by 5HT2C receptors. Neuropharmacology, 41, 200e209. Vigna, E., & Naldini, L. (2000). Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy. Journal of Gene Medicine, 2, 308e316. Virtanen, T., Pihlanto, A., Akkanen, S., & Korhonen, H. (2007). Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. Journal of Applied Microbiology, 102, 106e115. Werry, T. D., Loiacono, R., Sexton, P. M., & Christopoulos, A. (2008). RNA editing of the serotonin 5HT2C receptor and its effects on cell signalling, pharmacology and brain function. Pharmacology and Therapeutics, 119, 7e23. Zemel, M. B. (2005). The role of dairy foods in weight management. Journal of the American College of Nutrition, 24, 537Se546S.